Although current methods used to assess the normal and pathopysiology of Vitamin D metabolism have led to a greater understanding of the problem, present information is necessarily incomplete since direct measurement of all of the most significant compounds by a single technique in the plasma of both adults and children has never been accomplished. Accordingly, we propose to perfect a mass spectrometric method which is capable of quantitating the metabolites Vitamin D3 in a single plasma sample simply, directly, and routinely. We have synthesized D3, 25OHD3, l,25(OH)2D3, 24,25 (OH)2D3, 25,26(OH)2D3 and l,24,25(OH)3D3 with 3 deuteriums on the side chain and in the 26(27) position for internal standards to be added to plasma before extraction. After preliminary separation and purification by high performance liquid chromatography and glass capillary gas chromatography, each metabolite accompanied by its deuterated standard will be analyzed by mass spectrometry. The ratio of protium to deuterium in selected ion pairs of the spectrum will be used as an index of the amount of each metabolite originally present in the sample. Because the internal standard is the compound itself labelled with deuterium problems of unaccountable losses due to degradation or isomerization would be avoided and absolute chemical identification at the point of quantitation would be achieved. Application of this method could further understanding of the normal pathway of D metabolism and of those alterations which might occur in chronic renal insufficiency, hypophosphatemic Vitamin D resistant Rickets, osteomalacia due to anti-convulsant therapy or malabsorption syndromes and other diseases with a D component.